Rapid DNA chemical ligation for amplification of RNA and DNA signal
Rapid DNA chemical ligation for amplification of RNA and DNA signal
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DOI：
10.1021/bc700244s
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发表时间：
2008-01-01
影响因子：
4.7
通讯作者：
Ito, Yoshihiro
中科院分区：
文献类型：
--
作者：
Abe, Hiroshi;Kondo, Yuko;Ito, Yoshihiro
酶连接方法可用于DNA序列的诊断检测。在这里，我们描述了非酶硫代磷酸-碘乙酰DNA化学连接作为一种检测和鉴定RNA和DNA的方法的调查。DNA靶标上连接的特异性显示允许区分单个点突变，连接产率下降高达16.1倍。虽然酶促连接对RNA靶具有非常低的活性，但该反应对RNA靶非常有效。与RNA靶的化学连接的速度在5秒内达到70%的产率，这等于或优于连接酶介导的与DNA靶的连接。在短至100-120分钟的热循环下，该反应还表现出显著水平的信号放大，其中RNA或DNA靶标以催化方式作用以连接多对探针。
Enzymatic ligation methods are useful in the diagnostic detection of DNA sequences. Here, we describe the investigation of nonenzymatic phosphorothioate-iodoacetyl DNA chemical ligation as a method for the detection and identification of RNA and DNA. The specificity of ligation on the DNA target is shown to allow the discrimination of a single point mutation with a drop in the ligation yield of up to 16.1-fold. Although enzymatic ligation has very low activity for RNA targets, this reaction is very efficient for RNA targets. The speed of the chemical ligation with an RNA target achieves a 70% yield in 5 s, which is equal to or better than that of ligase-enzyme-mediated ligation with a DNA target. The reaction also exhibits a significant level of signal amplification under thermal cycling in periods as short as 100-120 min, with the RNA or DNA target acting in a catalytic way to ligate multiple pairs of probes.